Transcriptional enhancers have emerged as predominant functional elements in the noncoding portion of the human genome. Mounting evidence supports the existence of hundreds of thousands of enhancers in our DNA, far outnumbering the ~20,000 protein-coding genes. The fundamental importance of enhancers in humans becomes increasingly appreciated as human genetic studies implicate rapidly growing lists of noncoding sequence variants in human disease, of which many are likely to alter enhancer functions. Over the past two funding cycles of this R01, our group has been a leader in identifying and characterizing enhancers that are active in vivo in developmental and disease-relevant tissues, including developing and implementing strategies for enhancer discovery in the human and mouse genomes and building large catalogues of in vivo enhancers characterized in transgenic mouse assays. A fundamental finding from these studies is the complex nature of in vivo enhancer activity in time and space, which is not readily captured in cell lines, requiring studies directly of human or mouse tissues. Despite substantial progress in annotation of enhancers in the human genome, significant questions regarding distal enhancers remain, including: (1) How essential are enhancers in organismal function? (2) Which genes does each of these distant-acting enhancers regulate? (3) What differences in enhancer landscape exist among different cell types located within the same heterogeneous organ or tissue? (4) How can we enable and facilitate the discovery of mechanistic links between enhancers and human disease? Accordingly, in this competitive renewal, we propose to: (1) Use the exciting Cas9/CRISPR technology in conjunction with our established pipeline for large-scale mouse oocyte injections to efficiently and cost-effectively generate a series of enhancer knockout mice to assess enhancer function in vivo, (2) capture genome-wide enhancer-promoter interactions directly from mouse tissues to experimentally determine which genes specific enhancers regulate in vivo, (3) develop technology to use individual enhancers with well-defined activity patterns as drivers to isolate pure populations of labeled cells from transgenic mouse tissues, which will enable enhancer characterization at cell type-specific resolution, (4) continue to provide community access to genomic enhancer resources including high-throughput transgenics and new streamlined Cas9-engineered enhancer knockout capabilities to define enhancer function in vivo and assess the functional significance of variants linked to human disease. Through these studies, we expect to gain significant insight into the role of enhancers in mammalian biology, revealing the impact of enhancer deletions on gene expression and downstream phenotypes, the regulatory targets of enhancers, and the cell type specificity of enhancer activity within mammalian tissues. With the projected growth of clinical whole-genome sequencing, this work will be vital to aid in understanding and predicting the consequences of sequence variation in noncoding elements in human physiology and disease.